Compositions and methods for activating genes of interest

ABSTRACT

Compositions and methods for activating genes of interest are provided. The compositions comprise a masked targeted expression cassette which expresses a gene product only in the presence of a target molecule. The cassettes are useful for the treatment of disease and for preventing the proliferation of neoplastic cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/956998, filed Sep. 20, 2001; which is a continuation of U.S.application Ser. No. 09/446,402, now U.S. Pat. No. 6,323,003, filed Dec.20, 1999, which was a National Phase Entry of PCT/US98/13093, filed Jun.24, 1998, which National Phase Entry claimed priority from U.S.Provisional Application No. 60/050,772, filed Jun. 25, 1997, all ofwhich are herewith incorporated in their entirety by reference herein.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for activatinggenes of interest particularly in the presence of a target gene.

BACKGROUND OF THE INVENTION

The nature of and basic approaches to cancer treatment are constantlychanging. At present, adjuvant chemotherapy routinely follows localtreatment of cancers. Clinical protocols are now exploring genetictherapies, manipulations of the immune system, stimulation of normalhematopoietic elements, induction of differentiation in tumor tissues,and inhibition of angiogenesis. Research in these new areas has led toapplications for nonmalignant disease.

At the same time, the new clinical protocols have a narrow therapeuticindex as well as a great potential for causing harmful side effects. Athorough understanding of the pharmacology, drug interactions, andclinical pharmacokinetics is essential for safe and effective use inhuman beings.

The therapy of viral infection is in its infancy. Bacterial infection istypically treated with agents, such as antibiotics, which take advantageof the differences in metabolism between the infecting organism and itshost. However, viruses largely employed the host's own enzymes to effectthe replication, and thus leave few opportunities for pharmacologicalintervention. By employing strong regulatory elements, the virus obtainstranscription and translation of its own genes at the expense of hostgenes.

In mammals, viral infection is combated naturally by cytotoxicT-lymphocytes, which recognize viral proteins when expressed on thesurface of host cells, and lyse the infected cells. Destruction of theinfected cell prevents the further replication of the virus. Otherdefenses include the expression of interferon, which inhibits proteinsynthesis and viral budding, and expression of antibodies, which removefree viral particles from body fluids. However, induction of thesenatural mechanisms require exposure of the viral proteins to the immunesystem. Many viruses exhibit a dormant or latent phase, during whichlittle or no protein synthesis is conducted. The viral infection isessentially invisible to the immune system during such phases.

Retroviruses carry the infectious form of their genome in the form of astrand of RNA. Upon infection, the RNA genome is reverse-transcribedinto DNA, and is typically then integrated into the host's chromosomalDNA at a random site. On occasion integration occurs at a site whichtruncates a gene encoding an essential cellular receptor or growthfactor, or which places such a gene under control of the strong viralcis-acting regulatory element, which may result in transformation of thecell into a malignant state.

Viruses may also be oncogenic due to the action of their trans-actingregulatory factors on host cell regulatory sequences. In fact,oncogenesis was the characteristic which led to the discovery of thefirst known retroviruses to infect humans. HTLV-I and HTLV-II (humanT-lymphotrophic viruses I and II) were identified in the blood cells ofpatients suffering from adult T-cell leukemia (ATL), and are believed toinduce neoplastic transformation by the action of their transactivatingfactors on lymphocyte promoter regions. Two additional retroviruses havebeen found to infect humans. These viruses, HIV-I and HIV-II, are theetiological agents AIDS.

Current therapy for HIV infection includes new drugs called proteaseinhibitors. These drugs can dramatically reduce HIV levels in the bloodwhen taken with other antiviral compounds such as AZT. At the same time,natural weapons in the immune system's defenses polypeptide moleculescalled chemokines, have been unveiled as potent foes of HIV.

Antisense oligodeoxynucleotides have been proposed as a major class ofnew pharmaceuticals. In general, antisense refers to the use of small,synthetic oligonucleotides resembling single-stranded DNA, to inhibitgene expression. Gene expression is inhibited through hybridization tocoding (sense) sequences in a specific messenger RNA (mRNA) target byWatson-Crick base pairing in which adenosine and thymidine or guanosineand cytidine interact through hydrogen bonding.

Following the simple base-pairing rules which govern the interactionbetween the antisense oligodeoxynucleotides and the cellular RNA, allowthe design of molecules to target any gene of a known sequence. A majoradvantage of this strategy is the potential specificity of action. Inprincipal, an antisense molecule can be designed to target any singlegene within the entire human genome, potentially creating specifictherapeutics for any disease in which the causative gene is known. As aresult, there have been numerous applications of antisenseoligodeoxynucleotide (ODN) activity for potential antiviral andanticancer applications.

Antisense ODNs offer the potential to block the expression of specificgenes within cells. Despite numerous reports of apparent antisenseinhibition of gene expression in cultured cells, only in a few cases hasspecific inhibition been rigorously demonstrated. In many studies,specificity has been inferred from the biological effects of antisenseas compared to control ODNs, without measuring levels of target RNA orproteins to evaluate specificity. Unintended side-effects of antisensetechnology could potentially occur through a number of mechanisms.

The potential of oligonucleotides as modulators of gene expression iscurrently under intense investigation. Most of the efforts are focusedon inhibiting the expression of targeted genes such as oncogenes orviral genes. The oligonucleotides are directed either against RNA(antisense oligonucleotides) or against DNA where they form triplexstructures inhibiting transcription by RNA polymerase II. To achieve adesired effect, the oligonucleotides must promote a decay of thepreexisting, undesirable protein by effectively preventing its formationde novo.

There is therefore a need for the development of new antisense methodsthat are more potent, reliable and specific than those used in previousstudies.

SUMMARY OF THE INVENTION

Compositions and methods for activating the expression of a gene ofinterest is provided. The compositions are antisense masked expressioncassettes which comprise a double stranded nucleotide sequence. A firststrand comprises an armed expression cassette, i.e., an RNA moleculewhich codes for a protein of interest linked downstream of a flankingsequence and a translation initiation site operably inserted upstream ofthe RNA sequence. The flanking sequence encodes a target molecule. Thatis, the flanking sequence encodes a target gene or codes for RNA ofinterest. The flanking sequence corresponds to the “sense” strand of thetarget. A second nucleotide strand is also provided, capable ofhybridizing to the flanking sequence of the first nucleotide sequence;i.e., the antisense strand. The antisense strand masks the translationinitiation site when bound. The flanking sequence can be designed sothat the antisense sequences do not share 100% homology with theflanking sequence. Thus, in the presence of a target nucleotidemolecule, the antisense strand will favor complementary binding with thetarget. In this manner, the antisense strand will disassociate from thearmed strand and pair with the target. Disassociation of the antisensestrand unmasks the ribosome binding site allowing the armed cassette tobe translated in the presence of the target.

The compositions find use in regulation of gene expression, treatment ofdisease, and for preventing the proliferation of neoplastic cells.Additionally, the compositions have a broad range of use in both plantand animal applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a diagrammatic sketch of the masked targeted expressioncassette as an antiviral drug.

FIG. 2 provides a diagrammatic sketch of the masked targeted expressioncassette in which the target sequence of the sense strand is completelycomplementary to the antisense strand.

FIG. 3 provides a diagrammatic sketch of the masked expression cassettewith concatenated geometry for increasing target specificity.

FIG. 4 provides a diagrammatic sketch of the masked targeted expressioncassette with concatenated geometry which requires a quantity thresholdof target molecules for initiation of translation of the desired gene.

FIG. 5 provides a diagrammatic sketch of a circular masked targetedexpression cassette for increased compactness and decreased viscosity.

FIG. 6 provides a diagrammatic sketch of a stem-loop masked targetedexpression system for increased compactness.

FIG. 7 provides an example of a construct for production of the sensestrand of the targeted cassette. The Kozak sequence is also shown (SEQID NO:17).

FIG. 8 provides a diagrammatic sketch of an in vitro experimentutilizing the masked targeted expression cassette.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to preferred embodiments. This invention may, however, beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will convey the scope of the inventionto those skilled in the art.

Compositions and methods for controlling the expression of a gene ofinterest is provided. Expression is regulated by the use of antisenseoligonucleotides to a target molecule. In this manner, the gene ofinterest is expressed only in the presence of RNA or DNA correspondingto the target molecule.

The method involves the use of an antisense masked expression cassette.By antisense masked expression cassette is intended a double strandednucleic acid molecule. The first strand comprises an RNA molecule forthe protein of interest linked downstream of a flanking sequence. Theflanking sequence comprises a nucleotide sequence, the sense sequence,for a portion of the target gene. The first strand also comprises atranslation initiation site downstream of the flanking sequence. It isrecognized that the site of insertion for the ribosome binding site mayvary. Optionally, a seven methyl guanine cap can be included tostabilize the molecule. See FIG. 1.

The second strand of the masked expression cassette comprises anantisense oligonucleotide corresponding to the target gene or sequence.That is, the antisense sequence is at least partially complementary tothe target sequence comprised by the flanking sequence. The antisenseoligonucleotide may be either an RNA molecule, a DNA molecule ormixtures thereof. The duplex formed by binding of the second antisensestrand to the corresponding flanking sequence of the first strandexcludes ribosomal scanning of the downstream sequences; including thetranslation initiation site, the sequence of interest, or both. Thus,translation and expression of the protein of interest is masked by thebinding of the antisense strand to the flanking sequence. Displacementof the antisense strand from the flanking sequence unmasks expressionand translation of the protein of interest.

The protein of interest will vary depending upon the use of thecomposition. For example, where the masked cassette is intended as amechanism to inhibit the growth of neoplastic cells, the protein ofinterest is selected from toxin proteins, cytokines, cell regulators, orthe like. Additionally, the RNA molecule may be non-coding RNA, such asRNA with RNAse activity.

A number of toxin proteins are known and can be used in the invention.These include ribosome inactivators, Pseudomonas exotoxin A (Chaudharyet al. (1990) J. Biol Chem 265:16303-16310); cell metabolism disruptors,such as ribonucleases (See, for example, Mariani et al. (1990) Nature347:737-741); Barnase toxin, a chimeric toxin derived from Pseudomonasexotoxin A and a ribonuclease (Prior et al. (1990), Cell 64:1017-1023);Pertussis (Accession M14378 M16494, Micosia et al. (1986) Proc. Nati.Acad. Sci. USA 83:4631-4635); cholera (Dams et al. (1991) Biochim.Biophys. Acta 1090:139-141); Diphtheria, ricin (Gelfand et al. EP0335476-A2); etc. Additionally, thymidine kinase from the herpessequence may be used as a toxin or effector molecule. Transcription in acell makes it susceptible to gancyclovir. Thus, the cells of interestcould be labeled and then destroyed in a two step system.

The masked cassette comprising the above described toxin sequences canbe used to target and destroy any cell, organ, organism or species; solong as a target sequence can be identified that is specific to thatcell, organ, organism or species. For example, the cassette can be usedto selectively target and eliminate vertebrate environmental pests. Suchpests include agricultural pests including foxes and rabbits inAustralia.

The cassette can be used to selectively destroy cells infected withviruses. In this aspect, the target sequence comprises a virus-specificsequence, while the protein of interest is a toxin as described above.

The masked cassette can be used to treat a variety of diseases. Suchdiseases include, but are not limited to, diseases involving anoveractive organ, such as a hyperactive thyroid. In this aspect, themasked cassette comprises a thyroid-specific target sequence and aprotein toxin as described above.

The cassette can be used to treat diseases involving a defective gene.In this aspect, the target sequence comprises the sequence of thedefective mRNA, while the sequence of interest comprises the sequence ofthe normal protein. The intended affect can be twofold. The binding ofthe antisense strand to the defective mRNA can shut down the productionof the defective protein, while expression of the sequence of interestresults in production of the normal protein.

The cassette can be used to produce a protein of interest in an organwhich lacks the protein. In this aspect, the target sequence comprisesan organ-specific sequence, while the sequence of interest comprises thesequence of the protein lacking in that organ.

The invention is also useful in an assay system to determine thepresence of a target molecule. In this instance the protein of interestwill be a reporter protein that is easily detected, for example, byeither a simple cytological stain or an enzyme assay. Such reportersequences include but are not limited to beta galactosidase,chloramphenicol acetyltransferase (CAT), glucurodinase (GUS), and thelike.

A translation initiation site is also included in the cassette. Suchsequences are known in the art and include the Kozak sequence. See, forexample, Kozak, Marilyn (1988) Mol. and Cell Biol., 8:2737-2744; Kozak,Marilyn (1991) J. Biol. Chem., 266:19867-19870; Kozak, Marilyn (1990)Proc Natl. Acad. Sci. USA, 87:8301-8305; Kozak, Marilyn (1989) J. CellBiol., 108:229-241; and the references cited therein. Such referencesare herein incorporated by reference.

The translation initiation site can be inserted upstream of the sequencecorresponding to the gene of interest. Kozak sequences can be designedthat can initiate translation in all three reading frames. See, forexample, Murphy and Efstratiadias (1987) Proc. Natl. Acad. Sci. USA,84:8277-8281. Generally, the Kozak sequence will comprise the consensussequence recognized for initiation in higher eukaryotes. Such consensussequence is GCCGCC_(G) ^(A)CCAUGG (SEQ ID NO:18). This consensussequence is repeated several times within the Kozak sequence to providefor the initiation of translation in all three reading frames.

The length of the Kozak sequence may vary. Generally, increasing thelength of the leader sequence enhances translation.

It is recognized that a prokaryotic translation initiation site may alsobe used when appropriate; for example, when targeting a prokaryote. Suchsequences include the Shine-Dalgarno sequence (UAAGGAGG (SEQ ID NO:19)),typically 5-10 bases upstream of the initiator AUG.

The flanking sequence comprises a sequence which corresponds to thetarget gene or sequence. That is, the flanking sequence comprises all ora part of the sense strand of the target molecule and can be RNA or DNA.By sense sequence is intended a sequence capable of hybridizing to theantisense portion capable of hybridizing to messenger RNA expressed bythe target when the target is a gene, or to a target RNA or DNAmolecule.

The flanking sequence may vary in length. It is recognized that thelength may vary depending on the length and abundance of the targetgene, and the specificity and affinity of the antisense portion for thetarget. While the length of the flanking sequence may vary, generally alength of about 10 to about 200 nucleotides, preferably about 20 toabout 150 nucleotides, more preferably about 40 to about 100 nucleotidescan be used.

The flanking sequence can be a naturally occurring or syntheticsequence. Where the sequence is synthetic, mismatch nucleotides can beincorporated into the structure to facilitate thermodynamic displacementof the antisense molecule by the target molecule. It is recognized thatif the translation initiation site is inserted within the flankingsequence, this sequence insertion will provide non-hybridizing sequencesand add to the decrease in homology between the flanking sequence andthe antisense oligonucleotide. While it is recognized that a homology ofup to 100% can be compatible with the intended displacement of theantisense strand from the flanking sequence, generally a homology ofless than 90% is intended, preferably about 75% homology, morepreferably about 65% homology.

A 7-methyl guanine (7MeG) cap is known to increase the efficiency oftranslation. Thus, such a 7-methyl guanine cap can be included on the 5′end of the flanking sequence. See, for example, Shatkin (1976) Cell,9:645-653; Malone et al. (1989) Proc. Natl. Acad. Sci. USA,86:6077-6081; Fuerst and Moss (1989) J. Mol. Biol., 206:333-348 andKozak (1991) Gene Expression, 1:117-125.

The antisense sequence of the expression cassette of the invention isconstructed to hybridize with a nucleotide sequence of interest. Suchnucleotide sequences of interest include messenger RNAs from targetgenes, viral RNAs or DNAs, and the like. The antisense strand isconstructed to be homologous to the target. Generally, such homologywill be greater than the homology exhibited by the antisense strand tothe flanking sequence. Thus, in the presence of the target molecule, theantisense strand is displaced from the flanking sequence of the cassetteand hybridizes with the target molecule. To enhance displacement, thecassette can be constructed such that the antisense sequence is longerthan the flanking sequence, allowing for a 3′ or 5′ non-paired overhangor “sticky end” to bind the target molecule. This sticky end willenhance displacement of the antisense oligonucleotide.

As discussed, the target molecule may vary. For treatment of malignantor neoplastic cell growth, the target molecule will correspond to anucleotide which is only expressed or present in the neoplastic cell. Inthis case, the sequence of interest of the expression cassette willencode a toxin protein which is expressed in the presence of the targetto kill the cell. The expression cassette could also encode a cytokineor interferon to fight neoplastic growth. In some instances, acombination of expression cassettes encoding different proteins may beprovided. The target molecule can be a gene. Numerous target genes areknown in the art. Such genes include c-myc, n-myc, c-myb c-abl, c-kit,c-mos, bcr-abl, bcl-2, retinoblastoma-1, p-53, GM-CSF, G-CSF, Ick,IGF-1, egr-1 (Krieg, ImmunoMethods 1, 191 (1992)); c-fes (Ferrari etal., Cell Growth Differ. 1, 543 (1990)); c-fms (Wu et al., Oncogene 5,873 (1990)); c-fos (Block et al., in (77). pp. 63-70); N-ras (Skorski etal., J. Exp. Med. 175, 743 (1992)); Ha-ras (Saison-Behmoaras et al.,EMBO J. MD., 1111 (1991)); B-myb (Arsura et al., Blood 79, 2708 (1992));CSF-1 (Birchenall-Roberts et al., J. Immunol. 145, 3290 (1990));Myeloblastin (Bories et al., Cell 59, 959 (1988)); Erythropoietin(Hermine et al., Blood 78, 2253 (1991)); MZF-I (Bavisotto et al., J.Exp. Med. 174, 1097 (1991)); mdrl (Rivoltini et al., Int. J. Cancer 46,727 (1990)); IGF-1 receptor (Porcu et al., Mol. Cell. Biol. 12, 5069(1992)); Growth hormone (Weingent et al., Endocrinology 128, 2053(1991)); EGR-1(Neyses et al., Biochem. Biophys. Res. Commun. 181, 22(1991)); G proteins (Supra (1992)); MHC-1# (Cambe et al., Anti-CancerDrug Des. 7, 341 (1992)); Angiotensinogen (Cook et al., Antisense Res.Dev. 2, 199 (1992); Myogenin (Brunetti et al., J. Biol. Chem. 265, 13435(1990)); LH receptor** (West et al., Mol. Cell. Endocrinol. 79, R9(1991)); Cellular retinol-binding protein I, (Cope et al., in (77), pp.125-142); TNF-α (A. Witsell and L. Schook, Proc. Natl. Acad. Sci. U.S.A.89, 4754 (1992)).

Target molecules include but are not limited to the CD4 gene, see,Accession No. X87579; CFTR gene (Varon et al. (1995) Hum. Mol. Genet4:1463-1464); human C3d/Epstein-Barr virus receptor (Fujisaku et al.(1989) J. Biol. Chem. 264:2118-2125); Human MHC class I CD8 alpha-chaingene (Accession M27161, Nakayama et al. (1989) Immunogenetics30:393-397); human elastase 2 mRNA (Accession M16631), Fletcher et al.(1987) Biochemistry 26:7256-7261); Human elastin mRNA (Accession M36860,Fazio et al. (1988) J. Invest. Dermatol. 91:458-464); humanintercellular adhesion molecule 1 gene (Accession U86814); humaninterleukin 1-beta converting enzyme isoform beta mRNA (Accession U13697Alnemri et al. (1998) J. Biol. Chem. 270:4312-4317); humanimmunoglobulin C mu-C delta locus (Accession X57331, Word et al. (1989)Int. Immunol 1:296-309); human interleukin 2 gene (Accession J00264,Maeda et al. (1983) Biochem. Biophys. Res. Commun. 115:1040-1047; Fujitaet al. (1983) Proc.

Natl. Acad. Sci. USA 80:7437-7441); human MHC Class I antigen HLA-B(Accession U88407); human NMC class II HLA-DPA1 antigen (AccessionU87556); etc. herein incorporated by reference.

Likewise, the target molecule may be a RNA or DNA from a virus. In thismanner, viral replication and growth can be inhibited. Such viral genesinclude but are not limited to sequences from Coxsackievirus (Marquardtand Ohlinger (1995) J. Virol. Methods 53:189-199); Dengue virus, seeAccession No. U88535; encephalitis virus, see, Accession No. AB001026;Ebola virus (Sanchez et al. (1989) Virology 170:81-91, Accession No.L11365); Epstein-Barr virus (Baer et al. (1984) Nature 310:207-211);Echovirus 32 (Huttunen et al. (1996) J. Gen. Virol. 77:715-725);Enterovirus (VP4-VP2 capsid 3D RNA polymerase genes Pulli et al. (1995)Virology 212:30-38); influenza A virus (Guan et al. (1996) J. Virol.70:8041-8046); hepatitis B virus (Fukuda et al. (1995) J. Infect. Dis.172:1191-1197); hepatitis C virus (Hitomi et al. (1995) Viral Immunol.8:109-119); hepatitis D virus (Khudyakov et al. (1993) Virus Res.27:13-24); hepatitis E virus (Tam et al. (1990) Science 247:1335-1449,Accession No. M32400); hepatitis G virus (Accession No. U86023); HIV(Accession U04908, Gao et al. (1996) J. Virol. 70:1651-1667); humanpapillomavirus (Accession U37537, Wu et al. (1993) Lancet 341:522-524);influenza A virus (Accession U86987); human rhinovirus (AccessionD00239, Hughes et al. (1988)J. Gen. Virol. 69:49-58); Sendai virus(Accession D00053 N00053, Morgan and Rakestraw (1986) Virology154:31-40); gastroenteritis virus TFI virion protein gene (AccessionZ35758; Chen et al. (1995) Virus Res. 38:83-89); herpes simplex type 2virus (Accession Z86099, McGeoch et al. (1987) J. Gen Virol. 68:19-38);Venezuelan equine encephalitis virus (Accession L01442, Kinney et al.(1986) Virology 152:400-413); herein incorporated by reference.

Other genes of interest include, for example, jun, bFGF, wnt-1,TGF-beta, spi- 1 for cytomegalovirus; NDR, c-erbB-2 for herpes simplexvirus, types 1 and 2; bcl-2 and bci-abl for human papilloma virus; p53and c-myb for hepatitis, type B; 1-myc and ras for influenza virus; etc.

Methods are generally available in the art for construction of themasked expression cassettes. See, for example, Sambrook et al., ColdSpring Harbor, N.Y. RNA/DNA molecules as well as antisenseoligonucleotides can be made in accordance with known techniques. See,e.g., U.S. Pat. No. 5,149,797; 5,175,273; Uhlmann and Peyman (1990)Chem. Rev., 90:543-584 and the references cited therein. The antisenseoligonucleotides, which may be deoxyribonucleotide or ribonucleotidesequences which are capable of complementary binding to the targetmolecule. Such antisense oligonucleotides may be oligonucleotideswherein at least one, or all, of the internucleotide bridging phosphateresidues are modified phosphates, such as methyl phosphonates, methylphosphonothioates, phosphoromorpholidates, phosphoropiperazidates andphosphoramidates. For example, some, for example, every other one, ofthe internucleotide bridging phosphate residues may be modified asdescribed. In another example, such antisense oligonucleotides areoligonucleotides wherein at least one, or all, of the nucleotidescontain a 2′ loweralkyl moiety (e.g., C1-C4, linear or branched,saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl,1-propenyl, 2-propenyl, and isopropyl). See also Furdon et al. (1989)Nucleic Acids Res., 17:9193-9204; Agrawal et al. (1990) Proc. Natl.Acad. Sci. USA, 87:1401-1405; Baker et al. (1990) Nucleic Acids Res.,18:3537-3543; Sproat et al. (1989) Nucleic Acids Res., 17:3373-3389;Walder and Walder (1988) Proc. Natl. Acad. Sci. USA, 85:5011-5015.

Modification of the phosphodiester backbone has been shown to impartstability and may allow for enhanced affinity and increased cellularpenetration of ODNs. Additionally, chemical strategies may be employedto replace the entire phosphodiester backbone with novel linkages.Phosphorothioate and methylphosphonate modified ODNs may be made throughautomated ODN synthesis.

A phosphorodithioate version of the phosphorothioate can be synthesized.In the dithioate linkage, the non-bridging oxygens can be substitutedwith sulfur. This linkage is highly nuclease resistant.

Sugar modifications may also be used to enhance stability and affinityof the molecules. The alpha-anomer of a 2′-deoxyribose sugar has thebase inverted with respect to the natural beta-anomer. ODNs containingalpha-anomer sugars are resistant to nuclease degradation.

If necessary, targeted cassette can be modified to increase stability invivo. Thus, nuclease-resistant oligonucleotides can be utilized, such asPS and MP oligonucleotides. See, for example, Miller (1991)Biotechnology, 9:358 and Stein et al. (1991) Pharmacol. Ther., 52:365.

The targeted expression cassettes of the invention can be synthesizedeasily and in bulk. The development of phosphoramidite chemistry and itselaboration into an automated technology have greatly enhanced the easewith which oligos are synthesized and consequently their availability.See, for example, Beaucage and Caruthers (1981) Tetrahedron Lett.,37:3557 and Zon and Geiser (1991) Anti-Cancer Drug Des., 6:539.

The methods, oligonucleotides and formulations of the present inventionhave a variety of uses. They are useful in preventing the proliferationand growth of neoplastic cells. The methods, oligonucleotides andcompositions of the present invention are also useful as therapeuticagents in the treatment of disease. They also find use in fermentationprocesses where it is desirable to have a means for regulating theexpression of a gene to be expressed at a certain time or any instancewhere it is desirable to regulate gene expression.

The term “antisense oligonucleotides” includes the physiologically andpharmaceutically acceptable salts thereof: i.e., salts that retain thedesired biological activity of the parent compound and do not impartundesired toxicological effects thereto. Examples of such salts are (a)salts formed with cations such as sodium, potassium, spermidine, etc.;(b) acid addition salts formed with inorganic acids, for examplehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid and the like; (c) salts formed with organic acids such as,for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid,polyglutamic acid, napthalenesulfonic acid, methanesulfonic acid,p-toluenesulfonic acid, napthalenedisulfonic acid, polygalacturonicacid, and the like; and (d) salts formed from elemental anions such aschlorine, bromine, and iodine.

Formulations of the present invention comprise the masked cassette in aphysiologically or pharmaceutically acceptable carrier, such as anaqueous carrier. Thus, formulations for use in the present inventioninclude, but are not limited to, those suitable for parenteraladministration, including subcutaneous, intradermal, intramuscular,intravenous and intra-arterial administration, as well as topicaladministration (i.e., administration of an aerosolized formulation ofrespirable particles to the lungs of a patient afflicted with cysticfibrosis). The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art.Such formulations are described in, for example, Remington'sPharmaceutical Sciences 19th ed., Osol, A. (ed.), Mack Easton Pa.(1980). The most suitable route of administration in any given case maydepend upon the subject, the nature and severity of the condition beingtreated, and the particular active compound which is being used.

The present invention provides for the use of the targeted maskedcassette having the characteristics set forth above for the preparationof a medicament for the various disorders. In the manufacture of amedicament according to the invention, the masked cassette is typicallyadmixed with, inter alia, an acceptable carrier. The carrier must, ofcourse, be acceptable in the sense of being compatible with any otheringredients in the formulation and must not be deleterious to thepatient. The carrier may be a solid or a liquid. One or more antisenseoligonucleotides may be incorporated in the formulations of theinvention, which may be prepared by any of the well known techniques ofpharmacy consisting essentially of admixing the components, optionallyincluding one or more accessory therapeutic ingredients.

Formulations of the present invention may comprise sterile aqueous andnon-aqueous injection solutions of the active compound, whichpreparations are preferably isotonic with the blood of intendedrecipient and essentially pyrogen free. These preparations may containanti-oxidants , buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient. Aqueousand non-aqueous sterile suspensions may include suspending agents andthickening agents. The formulations may be presented in unit dose ormulti-dose containers, for example sealed ampoules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, saline orwater-for-injection immediately prior to use.

In the formulation the targeted cassette may be contained within a lipidparticle or vesicle, such as a liposome or microcrystal, which may besuitable for parenteral administration. The particles may be of anysuitable structure, such as unilamellar or plurilamellar, so long as thetargeted cassette is contained therein. Positively charged lipids suchas N-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate,or “DOTAP,” are particularly preferred for such particles and vesicles.The preparation of such lipid particles is well known. See, e.g., U.S.Pat. Nos.4,880,635 to Janoff et al.; 4,906,477 to Kurono et al.;4,911,928 to Wallach; 4,917,951 to Wallach; 4,920,016 to Allen et al.;4,921,757 to Wheatley et al.; etc.

The dosage of the targeted cassette administered will depend upon theparticular method being carried out, and when it is being administeredto a subject, will depend on the disease, the condition of the subject,the particular formulation, the route of administration, etc. Ingeneral, intracellular concentrations of the cassette of from 0.05 to 50μM, or more particularly 0.2 to 5 μM, are desired. For administration toa subject such as a human, a dosage of from about 0.01, 0.1, or 1 mg/Kgup to 50, 100, or 150 mg/Kg is employed.

Current technology has focused on antisense molecules only. Antisenseoligonucleotides bind the offending RNA molecules in the cell. To beeffective, a high dosage of antisense molecules have to be delivered toeach cell. The present invention provides for an effector molecule whichincreases the potency of antisense technology. In this manner, the cellcan be manipulated more easily and a far lower dosage, potentially evena 1 molecule to 1 cell ratio can be effective.

It is the idea of specificity that provides the underlying feature ofthe present invention. Standard cytotoxic chemotherapy for conditionssuch as neoplastic disease is fraught with systemic toxicity. The ratioof the toxic dose to the therapeutic dose is relatively low, whichreflects the large number of cellular targets affected by thechemotherapeutic agent and the agents inability to distinguish betweennormal and diseased cells. In theory, this problem is solved by takingadvantage of the specificity conferred by Watson-Crick base pairformation by identifying an appropriate target.

The following experiments are offered by way of illustration and not byway of limitation.

EXPERIMENTAL

Use of Targeted Cassette to Kill Neoplastic Cells Following theprotocols as essentially described above, a targeted cassette isconstructed wherein the first strand has an RNA coding for toxin A. Thetoxin RNA is linked with upstream DNA sequences coding the sense portionof the p53 DNA sequence. The p53 protein is found in numerous cancercells. Inserted within the p53 molecule is a Kozak sequence. Anantisense structure is constructed which corresponds to the p53 sensenucleotide.

The targeted cassettes are provided to a patient in a pharmaceuticallyacceptable solution at a concentration of from about 0.01, 0.1, or 1mg/Kg up to 50, 100, or 150 mg/Kg.

Use of Targeted Cassette as Antiviral Drug

FIG. 1 depicts utilization of the masked targeted expression cassette asan antiviral agent (Black RNA Drug). Features of the cassette areidentified by the provided key. The sense strand has a 7meG cap at its5′ end. In the inactive form, the antisense strand is hybridized to theflanking sequences of the sense strand; such that the Kozak sequence ismasked. Mismatch area between the Kozak sequence and the antisensestrand is indicated by lack of hydrogen bonding. Upon presentation ofactive viral RNA which has perfect homology to the antisense strand, theantisense strand dissociates from the sense strand and binds the viralRNA, which renders the viral RNA inactive. Furthermore, upondissociation of the antisense strand, the Kozak sequence is unmasked andtranslation of the toxin protein commences from the AUG initiationcodon. Upon production of toxic quantities of mature toxin, the cellhosting the virus is destroyed.

Targeted Cassette with Totally Complementary Antisense and FlankSequences

FIG. 2 depicts a masked targeted expression cassette in which the viraltarget sequence of the sense strand is completely complementary to theantisense strand. In the inactive targeted cassette, ribosomal assemblyand scanning from the 5′ end is prevented by the duplex between theantisense strand and the flanking sequence. In this example,displacement of the antisense strand and activation of expression of thegene of interest (lac Z) can be tested by assaying for β-galactosidaseactivity.

Targeted Cassette with Increased Target Specificity

FIG. 3 depicts a masked targeted expression cassette with concatenatedgeometry for increasing target specificity for initiation of translationof the gene of interest. Each antisense/sense combination (1-3)corresponds to a different target sequence. For example, 1 correspondsto a viral RNA, 2 corresponds to a cytokine RNA. and 3 corresponds to ahost specific protein. RNA encoding the protein of interest is onlyexpressed when all 3 target sequences are present in the target cell,effecting displacement of all 3 antisense sequences from the sensesequences of the cassette, thereby allowing ribosomal assembly andscanning from the 5′ end to proceed to the Kozak sequence and the AUGstart codon.

Targeted Cassette with Target Quantity Threshold

FIG. 4 depicts a masked targeted expression cassette with concatenatedgeometry for requiring a target quantity threshold for initiation oftranslation. All sense/antisense combinations (1-3) correspond to thesame target RNA. RNA encoding the protein of interest is only expressedwhen the target RNA is present in sufficient quantity (in this example 3copies) in the target cell, effecting displacement of all 3 antisensesequences from the sense sequences of the cassette, thereby allowingribosomal assembly and scanning from the 5′ end to proceed to the Kozaksequence and the AUG start codon. The concatenated geometry thusrequires a threshold quantity of the target RNA for initiation oftranslation. Such concatenated cassette constructs are particularlyuseful for targeting cancer cells with abnormally high number of copiesof a particular mRNA. The constructs may also be made in combinationwith the constructs of FIG. 3.

Circular Targeted Cassette

FIG. 5 depicts a circular masked targeted expression cassette.

An antisense molecule complementary to a target molecule is bound tocomplementary sequences at the 3′ and 5′ end of the targeted expressioncassette, thereby preventing ribosome assembly and scanning from the 5′end. Thus, displacement of the antisense strand in the presence of acomplementary target molecule allows for translation and expression ofthe desired protein. The circular configuration may be more compact andless viscous, thereby having particularly desirable properties for drugdelivery applications.

Stem-loop Targeted Cassette

FIG. 6 depicts a stem-loop masked targeted expression cassette. Anantisense molecule complementary to a target molecule is bound tocomplementary sequences within the loop structure, further stabilizingthe stem-loop structure. Ribosomal scanning from the 5′ end is preventedfrom proceeding to the Kozak sequence and the initiation AUG codon bythe stable secondary structure of the complex. Displacement of theantisense strand in the presence of a complementary target moleculeprovides a less stable stem-loop structure unable to prevent ribosomalscanning commenced from the 5′ end.

Armed Sense Strand Plasmid Construct.

FIG. 7 depicts a pCI-Neo (Promega Corp.) plasmid construct forproduction of the sense RNA strand of a targeted expression cassette.The complete sequence of the depicted MCS-Kozak-lac Z is set forth inSEQ ID NO: 1. Alternative flanking sequences corresponding to portionsof the firefly luciferase mRNA are inserted into the multiple cloningsite (MCS), such that transcription from the T₇ promoter yields RNAcomprising from the 5′ end; luciferase segment-Kozak-β gal. Thealternative luciferase sense segments are set forth in SEQ ID NO: 2, 3,4, 5, 6, 7 or 8.

In Vitro Determination of Activity of Masked Targeted ExpressionCassette.

Sense strand RNA of the masked targeted cassette is produced by in vitrotranscription of the construct depicted in FIG. 7, by use of theRiboprobe® Combination System (Catalogue No. P1450, Promega Corp.).

Antisense sequences corresponding to portions of the target molecule(firefly luciferase RNA, Catalogue No. L4561, Promega Corp.) arehybridized to complementary flanking sequences of the sense strand ofthe targeted cassette. SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8 list alternativeflanking sequences and SEQ ID NO:9, 10, 11, 12, 13, 14 or 15 list thecorresponding antisense sequences, respectively. The full length fireflyluciferase RNA, according to which the flanking sense sequences andcorresponding antisense sequences are made is set forth in SEQ ID NO:16. The hybridized mixture is introduced to an in vitro translationmixture containing ribosomes and full length firefly luciferase RNA(Flex®, Rabbit Reticulocyte Lysate System, Catalogue No. L4540, PromegaCorp.). Control reactions will lack the masked cassette.

After completion of translation, the mixture will be assayed forP-galactosidase (β-gal) and luciferase activities. Negative luciferaseand positive β-gal activity indicates successful inhibition of thetarget molecule and successful expression of the gene of interest.

The diagram depicts the mechanism of the assay. In panel 1, theantisense sequence is bound to complementary flanking sequence of thetargeted cassette. Ribosomal scanning commenced from the 5′ end isblocked by the antisense/sense duplex, thereby preventing translation ofthe β-gal RNA. Displacement and binding of the antisense to targetluciferase RNA (panel 2) has a two-fold effect (panel 3). β-gal can beexpressed from the unmasked cassette (β-gal positive) and expression ofthe target is blocked by binding of the antisense to the target(luciferase negative).

Other modifications and embodiments of the invention will come to mindin one skilled in the art to which this invention pertains having thebenefit of the teachings presented herein. Therefore, it is to beunderstood that the invention is not to be limited to the specificembodiments disclosed. Although specific terms are employed, they areused in generic and descriptive sense only and not for purposes oflimitation, and that modifications and embodiments are intended to beincluded within the scope of the appended claims.

1-3. (canceled)
 4. A masked expression cassette comprising a doublestranded nucleic acid molecule comprising a first strand comprising a 5′sense sequence and a 3′ sense sequence; said first strand furthercomprising an RNA sequence which codes for a protein of interest linkeddownstream of said 5′ sense sequence, and a translation initiation siteoperably inserted upstream of the RNA sequence; and, a second strandbound to said 5′ sense sequence and to said 3′ sense sequence, whereinsaid second strand comprises a polynucleotide corresponding to said 5′sense sequence and to said 3′ sense sequence and to a target molecule.5. The cassette of claim 4, wherein said cassette further comprises a7-methyl guanine cap linked to the 5′ end of said nucleic acid molecule.6. The cassette of claim 5, wherein said protein of interest comprises atoxin selected from the group consisting of Pseudomonas exotoxin A,ribonuclease, Barnase toxin, Pertussis toxin, and cholera toxin. 7-12.(canceled)